Protective effects of chitosan based salicylic acid nanocomposite (CS-SA NCs) in grape (Vitis vinifera cv. ‘Sultana’) under salinity stress

Salinity is one of the most important abiotic stresses that reduce plant growth and performance by changing physiological and biochemical processes. In addition to improving the crop, using nanomaterials in agriculture can reduce the harmful effects of environmental stresses, particularly salinity. A factorial experiment was conducted in the form of a completely randomized design with two factors including salt stress at three levels (0, 50, and 100 mM NaCl) and chitosan-salicylic acid nanocomposite at three levels (0, 0.1, and 0.5 mM). The results showed reductions in chlorophylls (a, b, and total), carotenoids, and nutrient elements (excluding sodium) while proline, hydrogen peroxide, malondialdehyde, total soluble protein, soluble carbohydrate, total antioxidant, and antioxidant enzymes activity increased with treatment chitosan-salicylic acid nanocomposite (CS-SA NCs) under different level NaCl. Salinity stress reduced Fm', Fm, and Fv/Fm by damage to photosynthetic systems, but treatment with CS-SA NCs improved these indices during salinity stress. In stress-free conditions, applying the CS-SA NCs improved the grapes' physiological, biochemical, and nutrient elemental balance traits. CS-SA NCs at 0.5 mM had a better effect on the studied traits of grapes under salinity stress. The CS-SA nanoparticle is a biostimulant that can be effectively used to improve the grape plant yield under salinity stress.


Results
Chlorophyll fluorescence parameters. According to the results, chlorophyll fluorescence parameters were significantly affected by salinity stress and foliar application of CS-SA NCs at 1% and 5% probability ( Table 1). The salinity stress and foliar application along with the CS-SA NCs on the photosynthetically active radiation (PAR) value revealed that the foliar application in 0 and 50 mM salinity treatments did not significantly affect the PAR value. The highest PAR values were observed in 0.5 mM of CS-SA NCs under 100 mM salinity. Our findings indicated that salinity stress and the foliar application of the nanocomposite did not significantly influence the minimum fluorescence value (F 0 ). But, maximum fluorescence (Fm) decreased with increasing salinity stress and the mentioned treatments, so the highest Fm value was observed in the treatment with 0.5 mM of CS-SA NCs without salinity stress. Also, the application of CS-SA NCs (0.5 mM) at different salinity levels increased the electron transfer rate (ETR). Fm' decreased with enhancing salinity stress, but this reduction was significantly higher in unsprayed samples than in the plants treated by CS-SA NCs. Based on the obtained results, the highest maximum quantum efficiency of photosystem II (Fv/Fm) was observed in 0.5 mM of CS-SA NCs without salinity, and the lowest belonged to 100 mM salinity treatment and no foliar application (Table 1). www.nature.com/scientificreports/ Chlorophyll and carotenoid content. The results showed that chlorophyll a, b, and total, and carotenoids content were significantly influenced by salinity stress and foliar application of CS-SA NCs at 1% and 5% probability (Table 2). Based on the results of the comparison of means, the highest chlorophyll a content (22.8 mg/g FW) was measured in without salinity along with 0.5 mM of CS-SA NCs, and the lowest was recorded in grape plants under 100 mM salinity and without any foliar application. also chlorophyll b content reduced using salinity stress that the maximum obtainedat 0.5 mM of CS-SA NCs in stress-free conditions, and the lowest content was observed in 100 mM salinity treatment and no CS-SA NCs foliar application. The application of CS-SA NCs (0.5 mM) at different salinity levels significantly increased the total chlorophyll compared to the other treatments. The foliar application significantly increased leaf carotenoid content and the highest was obtained from the plants sprayed with 0.5 mM of CS-SA NCs and stress-free conditions (Fig. 1).
Osmolytes and membrane stability. Based to the ANOVA, salinity stress and foliar application of CS-SA NCs had a significant impact on proline, MDA, carbohydrate, and electrolytes at 1% probability, while the effects were not significant on protein content ( Table 3). The results showed that leaf proline content enhanced with increasing salinity stress. The 100 mM salinity and 0.5 mM of CS-SA NCs foliar application contained the highest proline content, and the lowest was observed in the control plants. Lipid peroxidation of the membrane boosted under salinity stress. The highest content of MDA belonged to the grape plants supplemented with 100 mM salinity and no foliar application treatments, and the lowest amount was found in the control. Increasing the concentration of CS-SA NCs foliar treatment improved the total soluble protein content. Electrolyte leakage increased with increasing salinity stress. Application of 0.5 mM CS-SA NCs caused a significant reduction in electrolyte leakage at 100 mM NaCl salt stress. Application of CS-SA NCs under salt stress increased the soluble carbohydrates in grape plants (Fig. 2).

Biochemical parameters.
According to the findings, H2O2, enzyme antioxidant activity, and total antioxidant activity were significantly affected by salinity stress and foliar application of CS-SA NCs at 1% and 5% probability (Table 4). H 2 O 2 levels were maximized at 100 mM salinity level without foliar application, and the lowest belonged to the control treatment. The GPX activity increased in the plants subjected to salinity stress, www.nature.com/scientificreports/ and the uppermost was observed in 100 mM salinity stress along with 0.5 mM of CS-SA NCs. The increasing salinity level led to a significant enhancement in SOD activity, and it increased at 0.5 and 0.1 mM of CS-SA NCs compared to the control treatment. Also, the APX activity showed a significant increase in plants treated with salinity stress, so that the utmost activity was showed in 0.5 mM of CS-SA NCs (Fig. 3).
Nutrients content. Based to the results, N, P, K, Mg, Zn, Fe content, and Na + /K + were significantly modified by salinity stress and foliar application of CS-SA NCs at 1% and 5% probability ( Table 5). According to the findings, the N content declined significantly with increasing salinity levels, while CS-SA NCs foliar application ameliorated it at the different salinity levels. The P content lessedd significantly with increasing salinity stress, and the maximum content belonged to 0.5 mM of the CS-SA NCs treatment without salinity; the 100 mM NaCl without foliar application contained the lowest P level. The results revealed that the K content was minimal in 100 mM salinity treatment without CS-SA NCs foliar application, and the highest K level was obtained at 0.5 mM of CS-SA NCs without NaCl treatment. The lowermost of Mg content was recordedin the 100 mM salinity treatment without foliar application, which reduced with enhancing the salinity concentration. With increasing salinity stress, the Fe content showed a significant reduction in the lowest level in 100 mM of NaCl stress. The    www.nature.com/scientificreports/ Zn content decreased significantly in the grape plants exposed to salinity stress. The application of CS-SA NCs at 0.5 mM could effectively improve the Zn content of the grape leaves under salinity stress. At different levels of salinity stress, the Na + /K + ratio declined significantly in the plants treated with the CS-SA NCs foliar application concentrations ( Table 5). The salinity-CS-SA NCs interaction was not statistically significant on the Na + content. The data showed that the Na + content increased significantly with increasing salinity levels compared to the control treatment. Na + content showed a significant decrease in 0.5 mM of CS-SA NCs compared to the control treatment (Fig. 4).   , proline, total soluble protein, and total soluble carbohydrate with N, P, K, and Mg content, but these traits positively correlated with Na, Na/k, APX, SOD, CAT, GPX, DPPH, Fm' , ETR and PAR. A negative correlation was observed between Na and Na/K and other evaluated nutrients. Heat map analysis based on the response of the plants to salinity and CS-SA NCs foliar application revealed that the traits including MDA, H 2 O 2 , EL, Na, Na/K, SOD, APX, PAR, ETR, DPPH, CAT, GPX, proline, total soluble protein, and total soluble carbohydrate had positive accordance to salinity stress, and on the other hand, the evaluated nutrient content, photosynthesis pigment, and fluorescence chlorophyll decreased with increasing salinity stress. The heat map analysis showed CS-SA NCs foliar application recovered the adverse effect of salinity stress by improving the physiological and nutritional traits (Fig. 6).

Multivariate analysis of
Cluster analysis and dendrograms in the heat map ( Fig. 6) showed three main clusters in the evaluated features of the plants under salinity stress and CS-SA NCs foliar application. Cluster I comprised MDA, H 2 O 2 , EL, Na, Na/K, SOD, APX, PAR, ETR, DPPH, CAT, GPX, proline, total soluble protein and total soluble carbohydrate; cluster II comprised nutrients content and Fm; cluster Ш included photosynthesis pigments, Fm' and Fv/Fm (Fig. 7). In general, cluster analysis of heat maps for salinity stress combined with CS-SA NCs indicated three classes. Class I contained the plants under 50 and 100 mM of NaCl with 0.5 mM foliar application of CS-SA NCs; Class II contained the plants treated with 50 and 100 mM of NaCl with 0.1 mM Cs-SA NCs foliar application, as well as the raised plants under 50 and 100 mM of NaCl with no-foliar application. Finally, class III included the Table 5. Mean comparisons for the effects of CS-SA NCs under salinity on the nutrient element content of grapevine cv. 'Sultana' . Similar letters show no meaningful difference at 5% probability level by Duncan's Multiple Range Test. Data are mean ± SD (n = 3 replicates). ns, ** and *: Non-significant, significant at 1 and 5 percentage probability levels, respectively.   (Fig. 6).

Discussion
The measurement of chlorophyll fluorescence is one of the important, simple, and non-destructive methods to evaluate photosynthetic efficiency. Plant response to salinity depends on the ability of PSII to respond to salinity stress 29 . Salinity reduces the quantum yield of the PSII electron transfer, the amount of light energy reaching the reaction center, and the complex involvement of oxygen. When plastoquinone (PQ) is oxidized under natural conditions, the electron transfer system has a minimum value of Fo. In salinity stress, however, Fo increases due to changes in the structure of the thylakoid membrane and damage to the PSII reaction centers 30,31 . The Fv/Fm index indicates the initial yield of photosystem II and acts as a stress indicator as it is sensitive to early plant responses to stress 32 . A decrease in the Fv/Fm index was reported in wheat under salinity stress 33 . Under stress conditions, reductions in Fm, Fv, and Fv/Fm can inhibit electron transfer from the PSII reaction center to electron transfer 34 . In sweet pepper, salinity stress influenced chlorophyll fluorescence parameters and caused a significant reduction in the maximum PSII yield (Fv/Fm). The useful role of chitosan was reported in increasing www.nature.com/scientificreports/ the production of protective metabolites, increasing the contents of N and K as well as the number of chloroplasts under stress, thereby improving the chlorophyll fluorescence parameter 35 . In a study of salinity stress on strawberry 32 and sweet pepper 35 induction of chlorophyll fluorescence of leaves increased with increasing salinity levels. Also, NPQ and F 0 increased with increasing stress, but Fv/Fm and Fm decreased, which was consistent with our findings.  www.nature.com/scientificreports/ Based on the present results, a decrease in chlorophyll content was observed under salinity stress. Reductions of photosynthetic pigments under salinity stress may be caused by the deficiency in the leaf area responsible for light absorption and photosynthesis or may be due to chlorophyll degradation by increasing the activity of chlorophyll-degrading enzymes under salinity stress 36,37 . Other reasons for the reduction of photosynthetic pigments in salinity conditions include various types of ROS that cause chlorophyll degradation and damage to photosynthetic pigments 8 . According to our results, an increase in carotenoids with SA application was reported in tomato 38 , strawberry 39 , and myrtle (Catharanthus) 40 . Chitosan foliar application reduced the adverse effect of salinity stress by increasing the chlorophyll content. This increase was attributed to improvements in stomatal conductance, transpiration rate, and cell size and number 41,42 . Chitosan improved leaf chlorophyll content due to a higher nitrogen uptake, its transfer to leaves, and thus increasing chlorophyll pigments 42 . An increase in leaf chlorophyll content with the use of chitosan was reported in tomatoes 43 and cucumber 44 . Our results in chlorophyll content under stress and chitosan treatment were similar to the results of other researchers [43][44][45][46] .
Proline protects cells by improving osmotic regulation, inhibiting ROS increase, and protecting the membrane structure 47 . Proline plays an essential role as an osmotic stabilizer as well as a stabilizer and protector of enzymes, proteins, and membranes 48 . An increase in proline content is an indicator of stress reduction 47 . In a study on salinity stress in tomatoes, proline content increased due to its role in the regulation and inhibition of ROS 43 . Similar to the results of our research, an increase in proline content was reported in chitosan-treated tomatoes 43 and sunflower 47 under salinity stress conditions. In this study, an increase in proline was observed in salicylic acid and chitosan treatments, which was consistent with the results of other researchers 43,47,49 . An increase in lipid degradation rate and MDA production was observed under stress conditions with the formation of ROS, leading to cell damage and destruction 43 , which corresponds to the present results. SA application in salinity stress reduced the amount of MDA in tomatoes 50 . Chitosan pretreatment under salinity stress increased the activity of antioxidant enzymes and reduced MDA levels and the negative effects of salinity stress 43,51 . SA application in stress conditions leads to the expression of genes in plants that produce proteins that activate signaling pathways and, ultimately, programmed cell death 52 . SA is reported to stimulate the synthesis of stress-related proteins by increasing nitrate reductase activity 53 and increasing the content of abscisic acid 54 . Chitosan treatment also increased the content of total soluble proteins due to its role in increasing the expression of enzymes involved in glycolysis 55,56 . The increase in the production of malondialdehyde and decrease in degradation of lipids with the application of nano-chitosan-salicylic acid in the present results were consistent with the findings of other researchers 43,50 .
The activity of antioxidant enzymes increases in plants exposed to salinity stress because some antioxidant enzymes should be active to maintain lower levels of ROS 43,57 . SA activates the antioxidant enzymes SOD and CAT in stressed plants 13 . In myrtle, SA treatment in salinity stress increased the activity of SOD 58 . Chitosan can activate ROS-inhibitory systems in plants 43 . The use of chitosan as a bioelicitor with the potential to inhibit ROS has been shown in numerous studies. The activity of antioxidant enzymes increased significantly under the effect of chitosan treatment 59 . Chitosan could reportedly increase the activity of SOD and other antioxidant enzymes and cause tissue protection and delayed aging in stressed grapes 60 . Based on the results of the present study, the activity of superoxide dismutase and ascorbate peroxidase enzymes has increased significantly with increasing salinity and the application of salicylic acid-chitosan nanocomposite, which was consistent with the findings of other researchers in apple 61 , tomato 43 . The guaiacol peroxidase (GPX) enzyme is active in the cytosol, and glutathione is used as its cofactor 55 . This enzyme is present in the cellular and apoplasmic systems and is involved in many growth and development processes in the plant. Increasing salinity levels increased the activity of GPX 13 . The use of chitosan increased the activities of SOD, POX, and CAT in wheat and maize seedlings under salinity stress 62 . In the study of salt stress in spinach 63 , the activity of two enzymes, ascorbate peroxidase, and guaiacol peroxidase, increased significantly, which was consistent with our findings in grapes under salt stress.
NaCl salinity stress reduced the concentrations of Ca and Mg in all plant organs 64 . K uptake in salinity stress decreased significantly due to the effect of sodium on K transport in xylems and the inhibition of uptake processes 4 . Increased concentrations of Na + and Cl − because of salinity stress reduced the uptake of K + , Ca 2+ , and NO3 − and nutrient imbalance or deficiency 65 . In salinity conditions, a decrease in the Na + /K + ratio in the plant indicates its resistance to salinity stress 9,64 . SA affects the intracellular ion balance of Na + and K + by increasing the regulation of H + -ATPase activity and thereby increases plant salinity resistance 66 , which is in agreement with our results. Decreased uptake of K and Ca has been reported under high salinity levels. Osmotic damage in plants occurs due to high levels of Na in leaf apoplasts 7,57 . SA significantly increased Fe uptake in strawberries 39 and cucumber 67 . SA increases the amount of cytoplasmic K compared to Na by increasing the activity of the H + -ATPase pump in the cell membrane and providing a proton gradient, which contributes to reducing the toxic effects of Na and Cl and stimulates the activity of plant antioxidant systems and removal of ROS, thereby maintaining the integrity and protection of the cell membrane 68 . In the strawberry plant, leaf K content increased significantly by the effect of chitosan treatment 69 . Chitosan application in chickpea plants increased K content under salinity stress 70 . It seems that chitosan application caused the proper response of stressed grape plants to salinity stress by increasing and decreasing the concentrations of K and Na, respectively; in other words, chitosan could minimize ionic toxicity caused by salinity stress. In a study, the concentration of magnesium, calcium, potassium, iron and zinc elements decreased in the salinity stress of Selva strawberry 71 , which was consistent with our findings in grapes under salinity stress. At high levels of salinity, the ratio of sodium to potassium caused an ion imbalance, which is similar to the results of the present study, that salinity increased the ratio of sodium to potassium in cucumber plants 72 . In this study, salicylic acid treatment has reduced sodium ion concentration, which is in agreement with the findings of Jayakannan et al. 73 in Arabidopsis.

Methods
Plant material and treatments. The current research was carried out in 2021 in the research greenhouse of the Department of Horticultural Science and Engineering, Faculty of Agriculture, located at Maragheh University, with a geographic location of 46,16° N latitude and 22,37° E longitude. The homogeneous one-year-old rooted cuttings of Vitis vinifera L. cv. 'Sultana' was provided by the nursery of the Horticultural Science Department, University of Maragheh, Iran by the relevant institutional and national guidelines and legislations. They were cultured in 5 L pots containing soil with a loamy sand texture (Table 6). For the initial growth and the adaptation of grapevines to greenhouse conditions (16 and 8 h of light and darkness, 30:25 °C day and night temperature). During the growth period, necessary care such as irrigation and other operations was taken regularly.
After the full growth of the leaves of the seedlings, the treatments were carried out. To investigate the effects of foliar application of chitosan-salicylic acid nanocomposites (CS-SA NCs) on the physiological and biochemical properties of 'Sultana' cultivar grape in salinity conditions, a factorial experiment was used in completely randomized design (CRD) with three replications. One month after the establishment of the plants, salt stress was applied for one month, and during the stress period, the root environment of the plants was completely washed with salt-free water once every five days to minimize the changes in EC and pH due to washing, and nanocomposite foliar spraying Chitosan-salicylic acid was applied in two stages (the first stage two weeks after salt stress and the second stage at the end of salt stress). Treatments included: salinity stress at three levels (0, 50, and 100 mM NaCl) and foliar application of CS-SA NCs at three levels (0, 0.1, and 0.5 mM).

Synthesis of chitosan-salicylic acid nanocomposite (CS-SA NCs).
To prepare CS-SA NCs, a biopolymer was used to load salicylic acid. In this study, 0.1 g of low molecular weight nanocomposite powder (25 ml of 1 wt% acetic acid solution was added and stirred for 2 h at 70 °C using a magnetic stirrer to obtain a clear CS solution. 100 μl SA of the prepared solution was added to the CS solution, then stirred rapidly for 1 h Sodium tripolyphosphate (TPP) was used as a cross linker with a ratio of 2.5 to CS content. TPP was dissolved in 5 ml of distilled water and then slowly added to the CS-SA solution, then rinsed several times with distilled water to remove the reaction material from the supernatant. Figure   Superoxide dismutase (SOD) activity. SOD activity was determined by measuring the inhibition of light reduction of nitroblue tetrazolium at a wavelength of 560 nm. Doing this, 50 ml of 50 mM potassium phosphate buffer, pH: 7.5, was used. Then, 75 μM nitroblue tetrazolium, 13 μM methionine, 0.1 μM EDTA solution, and 4 μM riboflavin were added to the buffer and the solution was stored in a dark place 83 .

Chitosan-salicylic acid nanocomposite (CS-SA NCs) characterization.
Catalase (CAT) activity. 0.5 g of grape leaf samples were homogenized with 0.1 M cold potassium phosphate buffer (pH: 7.5) with 0.5 mM EDTA based on the method of Dezar et al. 84 . From the resulting supernatant, 0.05 ml was added to 1.5 ml of 0.1 mM phosphate buffer (pH: 7) and 1.45 ml of double distilled water. The reaction was started by adding 0.5 ml of 75 mM hydrogen peroxide and a decrease in adsorption was recorded at 240 nm for 1 min.
Leaves nutrient elements content. The flame photometric method (Corning, 410, England) was employed to measure the amount of sodium and potassium. The atomic absorption spectrometer (Corning, 410, England) was used to measure Zn, Fe, Ca, P, Mg, and Mn content (Corning, 410, England) and N content was quantified by Kjeldahl methods 85,86 .
The present experiment was performed as a factorial based on a completely randomized design with three replications. MSTATC (ver. 2.1, Michigan University), Minitab (ver. 17), and R software (ver. 3.6.3) were used for the ANOVA, cluster, biplot, and correlation analysis of data, respectively and, Excel (2016) was used to draw the figures. The means were compared using Duncan's multiple range tests at 5 and 1% probability levels.

Conclusion
The results of this research demonstrated that photosynthetic pigments decreased in 'Sultana' cultivar grape plants with increasing salinity stress, but it increased the content of osmolytes and antioxidant enzymes. Salinity interrupted ionic homeostasis and reduced nutrients. The application of the CS-SA NCs in stress and non-stress conditions positively affected the improvement of the studied traits of grape plants, improved nutrient levels, and reduced the Na level. Consequently, this nanocomposite represents an innovative approach that can be successfully used in grape plants to improve the yield under salinity stress. However, further validation is needed to determine their effectiveness in other plant species.

Data availability
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.